RAPID AND EFFICIENT ISOLATION OF TRANSFERRIN AND FERRITIN FROM MANDUCA-SEXTA

We report methods for the rapid purification of two iron-binding prote ins from larval hemolymph of Manduca sexta. Ferritin was purified in t wo steps by density gradient ultracentrifugation. To accomplish this, we utilized the relatively high level of ferritin present in the hemol ymph of this animal and augmented the density of the protein in vivo b y injection of iron sulfate. Nitrocellulose blots analyzed by laser de nsitometry showed hemolymph from iron-injected insects contained about 0.4 mg of ferritin per mi (approximately 0.7% of total hemolymph prot ein); of this, 62% was found as pure ferritin in the pellet formed dur ing ultracentrifugation. Following the density ultracentrifugation, we purified transferrin from the hemolymph subphase by immobilized metal ion affinity chromatography using a new gel, Novarose-SE1000/40 coupl ed to dipicolylamine (DPA) chelated with nickel. Higher capacity Ni(2)DPA-gel permitted good resolution of transferrin in the first chromat ography; a lower capacity of the same gel allowed purification of tran sferrin in a second step. Overall transferrin recovery was 52%. Larval hemolymph contained 0.770 mg transferrin/ml, representing about 1.3% of the total protein.